JP4241370B2 - Use of duckweed in high-speed, high-throughput screening - Google Patents

Abstract

Methods for high-throughput screening in duckweed are disclosed. In one aspect, these methods are used to identify nucleotide sequences encoding polypeptides of interest. In another aspect, these methods are used to identify nucleotide sequences that modulate the expression of a target nucleotide sequence. The methods combine the predictive benefits of screening in whole plants with the speed and efficiency of a high throughput system.

Description

  The present invention relates to a method for high-speed, high-throughput screening using duckweed.

  Duckweed is the only member of the monocot plant family Lemnaceae. All four genera and 34 species are small, floating, freshwater plants whose geographic extent spans the entire globe (Landolt, Biosystemic Investing on the Family of Duckweeds: The Family of Affinity in Lemnae Stiffung Rubel, Zurich (1986)). Duckweed is the most known morphologically degenerated plant, but most duckweed species have all the tissues and organs of larger plants including roots, stems, flowers, seeds and fronds . Duckweed species are well-studied and there is a substantial amount of literature detailing their ecology, taxonomy, life cycle, metabolism, susceptibility to diseases and pests, their reproductive biology, genetic structure and cell biology Exists. (Hillman, Bot. Review 27: 221 (1961); Landolt, Biosystemic Investigation on the Family of Stimulation of Duckweeds: The Family of Lemmaet-A Mant.

  The growth habit of duckweed is ideal for microbial culture methods. Plants grow through new foliar plant budding in a macroscopic manner similar to yeast asexual reproduction. This growth occurs by plant budding from dividing cells. The division region is small and is found on the ventral surface of the frond. Dividing cells are in two pockets on each side of the central vein. The small central vein region is also a site where roots are formed and a trunk is formed that connects each frond to its mother frond. The division pocket is protected by a tissue flap. The frond buds alternately from these pockets. The doubling time varies from species to species and is as short as 20 to 24 hours (Landalt, Ber. Schweiz. Bot. Ges. 67: 271 (1957); Chang et al., Bull. Inst. Chem. Acad. Sin. 24. : 19 (1977); Datko and Mudd, Plant Physiol. 65:16 (1970); Venkataraman et al., Z. Pflanzenphysiol. 62: 316 (1970)).

  Duckweed plants and duckweed nodule cultures can be efficiently transformed by any of a number of methods such as Agrobacterium-mediated gene transfer, ballistic bombardment, or electroporation. Stable duckweed transformation by transforming duckweed cells with both a nucleotide sequence of interest and a gene conferring resistance to the selective substance, followed by culturing the transformed cells in a medium containing the selective substance The body can be isolated. See US Pat. No. 6,040,498 and US Patent Application No. 60 / 221,705, which is hereby incorporated by reference.

  Recent advances in plant genome sequencing have provided a great deal of information regarding gene sequences encoding novel polypeptides and related nucleotide sequences that can be provided to control the expression of these coding sequences. However, these sequence data do not provide information on the exact role of these nucleotide sequences in physiologically important processes.

  In order to determine or confirm the function of new genes, it is generally necessary to express these genes in vivo. When screening a nucleotide sequence to determine its activity in plants, the highest success rate is predicted from a test for sequence activity in all plants. However, greenhouse testing of intact plants grown in soil takes time and space. A great deal of effort is required to prepare, care for, and score these screens, and these steps are generally not automatable.

  A screening method for nucleotide sequences in a system that combines whole plant screening, which is expected to be successful, with high-speed, high-throughput screening at reduced scale and cost is desirable. Accordingly, the present invention provides a method for performing high-speed, high-throughput screening in duckweed systems.

  Fast mass processing methods are provided for determining the biological function of nucleic acid molecules in duckweed systems and for identifying nucleic acid molecules of interest.

  The methods of the invention can be automated to allow rapid and efficient identification and characterization of nucleic acid molecules. The method can be performed in a high-density format so that multiple samples can be processed simultaneously. In addition, multiple steps of the method of the present invention can be performed in the same high density format. For example, in one embodiment, duckweed culture transformation, selection and regeneration can all be performed on the same high density format plate. After transformation of duckweed culture, a high speed screening of nucleotide sequences is provided by the method of the present invention so that the nucleic acid molecules of interest can be distinguished within a shortened time frame. Ultimately, a highly efficient recovery of transformed duckweed systems is provided by the method of the present invention.

  The method of the invention involves the transformation of duckweed plant culture, duckweed nodule culture, duckweed suspension culture or duckweed protoplast cell culture with nucleotide sequences. The duckweed system is then manipulated to identify the nucleic acid molecule of interest.

  In one embodiment, a duckweed plant culture, duckweed nodule culture, duckweed suspension culture or duckweed protoplast cell culture is transformed with a test nucleotide sequence and the biological function of the test nucleotide sequence is determined, To determine whether the test nucleotide sequence is a nucleic acid molecule of interest.

  In another embodiment, the duckweed plant culture, duckweed nodule culture, under conditions such that the reporter nucleotide sequence is incorporated into the DNA of the duckweed plant culture, duckweed nodule culture, duckweed suspension culture or duckweed protoplast cell culture. A duckweed, duckweed suspension culture or duckweed protoplast cell culture with a reporter nucleotide sequence and determine whether the reporter nucleotide sequence is incorporated into duckweed DNA so that it is operably linked to the nucleic acid molecule of interest To identify the nucleic acid molecule of interest.

  The present invention relates to a method for rapid high-throughput screening of nucleic acid molecules in duckweed systems for identifying and characterizing nucleic acid molecules of interest. These methods combine the screening of whole plants that are expected to be successful with the scale, effort, time and cost efficiency of high-throughput screening screens typically associated with single cell technology.

[Definition]
The method of the present invention is a high-speed, high-throughput screening method. As used herein, the “high-speed mass processing method” intends a method in which at least one of the following applies.

  (1) The method is carried out in a high density format, i.e., in such a way that one or more samples are processed simultaneously. For example, in certain embodiments, at least 12, or more, at least 24, at least 48, at least 96, or at least 384 or more samples can be treated in a single plate or other container. By adding a porous support system to each well, plates or other containers can be specifically adapted for the growth, maintenance, and automation of duckweed cultures. See, eg, US Provisional Patent Application No. 60 / 294,430, filed May 30, 2001, and US Patent Application entitled “Plates and Methods for High-Speed Mass Screening”, filed May 28, 2002. I want to be.

  (2) Implement the method so that multiple steps, such as transformation, selection and regeneration of duckweed cultures can be performed in the same high density format plate or vessel. In one embodiment, two or more steps selected from a transformation step, a selection step and a regeneration step are performed in the same plate so that transfer of duckweed culture is not required between steps.

  (3) Automation can be applied, i.e. one or more steps of the method can be carried out using a laboratory robot station.

  (4) The method of the present invention enables high-speed screening of nucleotide sequences. For example, in certain embodiments of the invention, only 4 to 16 weeks after transformation, eg, 4 weeks or less, 5 weeks or less, 6 weeks or less, 7 weeks or less, 8 weeks or less , 9 weeks or less, 10 weeks or less, 11 weeks or less, 12 weeks or less, 13 weeks or less, 14 weeks or less, 15 weeks or less, 16 weeks or less The converted duckweed can be assayed to identify the nucleic acid molecule of interest.

  (5) At least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65 transformed by the method of the present invention. % Or more, at least 70% or more, at least about 75%, at least about 80% or more, at least about 85% or more, at least about 90% or more, at least about 95% or more of a duckweed culture contains a test nucleotide sequence or a reporter nucleotide sequence A very efficient recovery of transformed duckweed lines is provided that results in transgenic duckweed lines.

  As used herein, the term “duckweed line” or “duckweed culture” includes duckweed plant cultures, duckweed nodule cultures, duckweed suspension cultures and duckweed protoplast cultures.

  As used herein, the term “duckweed plant culture” refers to a culture that contains mostly well-differentiated duckweed plants.

  As used herein, the term “duckweed nodule culture” means at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% Means a culture containing duckweed cells which are differentiated cells. As used herein, a “differentiated cell” is at least one phenotypic characteristic (eg characteristic cell morphology or marker nucleic acid or protein) that distinguishes it from undifferentiated cells or from cells found in another tissue type. Expression). In certain embodiments, the duckweed nodule culture comprises duckweed micronodule as described elsewhere herein.

  As used herein, the term “duckweed suspension culture” refers to a culture comprising dispersed duckweed cells, such as dispersed duckweed callus cells. Generally, duckweed suspension cultures contain both single cells and various sized unorganized cell aggregates.

  The term “duckweed protoplast culture” refers to a culture comprising duckweed cells in which at least about 50%, 60%, 70%, 80% or 90% duckweed cells lack a cell wall. Methods for generating protoplast cells from plant cells are described, for example, in Eriksson, Plant Protoplasts (1995), edited by Fowke et al., CRC Press (which is hereby incorporated by reference). .

  As used herein, the term “biological function” refers to a biological activity or property of a nucleic acid molecule or polypeptide. For example, the biological function of a nucleic acid molecule includes modulating a biological response, encoding a polypeptide, and modulating the expression of a target nucleotide sequence. Examples of biological functions of polypeptides include modulating biological responses, imparting structural properties of interest, imparting biochemical activities of interest, and imparting regulatory activities of interest and so on. In particular, non-limiting examples of regulatory and regulatory activities include the ability to bind to the substrate of interest, the ability to bind to the ligand of interest, the ability to catalyze the reaction of interest, the ability to modulate the response to plant hormones, plant growth regulators Ability to modulate responses to environmental disturbances, ability to modulate responses to physiological disturbances, ability to modulate responses to one or more pathogens, and to one or more toxins Such as the ability to adjust the response.

  As used herein, the term “expression” refers to the transcription or translation of a nucleotide sequence.

  The term “duckweed” refers to a member of the Lemnaceae family. This family is currently divided into the following four genera and 34 species of duckweed. The genus of Lemna (L. aequinocitalis, L. disperma, L. ecuadoriensis, L. gibbba, L. japonica, L. minor, L. miniscula, L. obscura, L. perpusilla, L. tenura, L. tenura, L. tenera, L. Spirodella (S. intermedia, S. polyrrhiza, S. puncata); Wolfia (Wa. Angusta, Wa. Anrrumiza, Wa. Australina, Wa. Boreales., Wa. Boreales.). Elongata, Wa.globosa, Wa.microscopi a, Wa.neglecta); and Wolfiella genus (Wl.caudata, Wl.denticulata, Wl.gladiata, Wl.hyalina, Wl.lingulata, Wl.repunda, Wl.rotunda and Wl.neotropica). If any other genus or species of Lemnaceae is present, it is also an aspect of the present invention. Landolt, Biosystemic Investigation on the Family of Duckweeds: Classify the Family of Lemnaceae-A Monograph Study GeH .

  As used herein with reference to a nucleotide sequence, “operably linked” means a plurality of nucleotide sequences arranged in a functional relationship with each other. For example, a promoter nucleotide sequence is operably linked to a second nucleotide sequence when the promoter nucleotide sequence is positioned so that transcription of the second nucleotide sequence can be induced.

  A method for identifying a nucleic acid molecule of interest includes transforming a duckweed plant culture, duckweed nodule culture, duckweed suspension culture or duckweed protoplast cell culture with a nucleotide sequence. In certain embodiments, the nucleotide sequence is a test nucleotide sequence or a reporter nucleotide sequence. Duckweed cultures can be transformed using any method known in the art. In one embodiment, one of the gene transfer methods disclosed in US Pat. No. 6,040,498 to Stump et al., Or US Patent Application No. 60/221705, which is hereby incorporated by reference. As a result, a stably transformed duckweed is obtained. The methods described in these references include gene transfer by ballistic bombardment using a particle gun coated with a nucleic acid containing a nucleotide sequence of interest (also known as gene gun bombardment, particle gun bombardment, or particle bombardment). Known)), gene transfer by electroporation, and Agrobacterium-mediated gene transfer including vectors containing the nucleotide sequence of interest. Selection and regeneration of transgenic duckweed lines is described in these references and elsewhere in this specification. In one embodiment, duckweed stably transformed by any one of the Agrobacterium-mediated methods disclosed in US Pat. No. 6,040,498 to Stomp et al. Is obtained. The Agrobacterium used is Agrobacterium tumefaciens or Agrobacterium rhizogenes. In another embodiment, duckweed cultures are transformed using PEG-mediated transformation. For example, Lazerri, Methods MoI. Biol. 49: 95-106 (1995); Mathur et al., Methods Mol. Biol. 82: 267-276 (1998); Detta et al., Methods Mol. Biol. 111: 335-347 (1999), which is hereby incorporated by reference.

  In one embodiment, a duckweed plant culture, duckweed nodule culture, duckweed suspension culture or duckweed protoplast cell culture is placed in a high density format plate or other container, such as a 24-well plate, 48-well plate, or 96-well plate. To transform. By adding a porous support to each well, the plate can be adapted to automate the processing of duckweed cultures. For example, US Provisional Patent Application No. 60 / 294,430, filed May 30, 2001, and US Patent Application entitled “Plates and Methods for High-Speed Mass Screening”, filed May 28, 2002 (cited) The contents of which are hereby incorporated by reference). In another embodiment, duckweed cultures are transformed in a single vessel and then duckweeds are transferred to a high density format. Duckweed can be manually transferred to a high density format, or the migration of duckweed plants, nodules, suspended cells can be automated using methods known in the art.

  The present invention provides a method for preparing duckweed nodule cultures that can be easily manipulated using automated procedures. The method of the present invention comprises grinding large duckweed nodules to produce duckweed “fine nodules” cultures. "Fine nodules" are duckweed nodules that are sufficiently small in size that can be sucked or dispersed using the nozzle of an automated liquid handler. In certain embodiments, the automated liquid handler or automated liquid handler pipette tip is modified to increase the size of the barrel and tip opening so that the fine nodules can move freely in and out of the pipette tip. Use any method, such as a mechanical method, an enzymatic method, or a method of growing duckweed under conditions that promote the formation of fine nodules, to pulverize duckweed nodules to produce duckweed "fine nodules" Can do. In one embodiment, a fine nodule culture is produced by extruding duckweed nodules through one or more layers of mesh screen. In a specific embodiment, a 30 mesh screen is used. The fine nodules can then be transferred to a high density format plate or other container. The fine nodule concentration can be adjusted so that most wells of the high density format plate or container have only one transformation event. The fine nodules of the present invention show foliar regeneration efficiency similar to that observed for duckweed nodules that have not been ground into strips.

  In certain embodiments of the invention, duckweed is transformed with a nucleic acid molecule comprising a test nucleotide sequence. In certain embodiments, the test nucleotide sequence comprises an expression cassette having a transcription initiation region operably linked to the test nucleotide sequence. A transcription initiation region (eg, a promoter) can be native, homologous, exogenous or heterologous to the host, or can be a naturally occurring sequence or a synthetically generated sequence. Exogenous is intended to mean that the transcription initiation region is not found in the wild-type host into which the transcription initiation region is introduced.

  Any suitable promoter known in the art can be used in accordance with the present invention (including bacterial, yeast, fungi, insect, mammalian and plant promoters). For example, a plant promoter including a duckweed promoter can be used. Examples of promoters include cauliflower mosaic virus 35S promoter, opain synthase promoter (eg, nos, mas, ocs, etc.), ubiquitin promoter, actin promoter, ribulose bisphosphate (RubP) carboxylase small subunit promoter, and alcohol dehydrogenase promoter. For example, but not limited to. The duckweed RubP carboxylase small subunit promoter is known in the art (Silverthrone et al., Plant Mol. Biol. 15:49 (1990)). Other promoters derived from viruses that infect plants, preferably duckweed, include promoters isolated from Dashen mosaic virus, Chlorella virus (eg Chlorella virus adenine methyltransferase promoter; Mitra et al., Plant Mol. Biol. 26). : 85 (1994)), tomato yellow gangrene virus, tobacco stem gangrene virus, tobacco gangrene virus, tobacco ring spot virus, tomato ring spot virus, cucumber mosaic virus, peanut stamp virus, alfalfa mosaic virus, Examples include, but are not limited to, sugar cane, basil reform, and badnavirus.

  Finally, a promoter can be selected to obtain the desired level of control. For example, in some instances it is advantageous to use a promoter that confers constitutive expression (eg, a manopine synthase promoter from Agrobacterium tumefaciens). Alternatively, in other situations, promoters that are activated in response to specific environmental stimuli (eg, heat shock gene promoter, desiccation-inducible gene promoter, pathogen-inducible gene promoter, wound-inducible gene promoter, and light-inducible gene promoter) or plant It is advantageous to use growth regulators such as promoters derived from genes induced by abscisic acid, auxin, cytokinin and gibberellic acid. As a further alternative, promoters that provide tissue specific expression can be selected (eg, root, leaf and flower specific promoters).

  The overall strength of a given promoter can be affected by the combination and spatial organization of cis-acting nucleotide sequences, such as upstream activation sequences. For example, an activated nucleotide sequence derived from the Agrobacterium tumefaciens octopine synthase gene can enhance transcription from the Agrobacterium tumefaciens manopine synthase promoter (US Patent No. No. 5,955,646). In the present invention, the expression cassette can contain an activated nucleotide sequence inserted upstream of the promoter sequence to enhance expression of the test nucleotide sequence. In one embodiment, the expression cassette is three of the Agrobacterium tumefaciens octopine synthase genes derived from the Agrobacterium tumefaciens manopain synthase gene operably linked to a promoter derived from the Agrobacterium tumefaciens manopine synthase gene. Includes upstream activation sequences (US Pat. No. 5,955,646, which is incorporated herein by reference).

  Expression cassettes can further include transcriptional and translational termination regions that are functional in plants. Any suitable termination sequence known in the art can be used in accordance with the present invention. The termination region may be from the same origin as the transcription initiation region, from the same origin as the nucleotide sequence of interest, or from another source. Convenient termination regions are available from the Agrobacterium tumefaciens Ti plasmids, such as the octopine synthase and nopaline synthase termination regions. Guerineau et al., Mol. Gen. Genet. 262: 141 (1991); Proudfoot, Cell 64: 671 (1991); Sanfacon et al., Genes Dev. 5: 141 (1991); Mogen et al., Plant Cell 2: 1261 (1990); Munroe et al., Gene 91: 151 (1990); Ballas et al., Nucleic Acids Res. 17: 7891 (1989); and Joshi et al., Nucleic Acids Res. 15: 9627 (1987). Examples of additional termination sequences are the pea RubP carboxylase small subunit termination sequence and the cauliflower mosaic virus 35S termination sequence. Other suitable termination sequences will be apparent to those skilled in the art.

  Expression cassettes containing one or more genes or nucleic acid sequences can be transferred and expressed in transformed plants. Thus, each nucleic acid sequence is operably linked to 5 'and 3' regulatory sequences. Alternatively, multiple expression cassettes can be provided.

  In general, an expression cassette contains a selectable marker gene for selecting transformed cells or tissues. Selectable marker genes include genes encoding antibiotic resistance such as genes encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), and genes conferring resistance to herbicidal compounds. . The herbicide resistance gene generally encodes a modified target protein that is insensitive to the herbicide or an enzyme that degrades or detoxifies the herbicide in the plant before the herbicide can act. DeBlock et al., EMBO J. et al. 6: 2513 (1987); DeBlock et al., Plant Physiol. 91: 691 (1989); Fromm et al., BioTechnology 8: 833 (1990); Gordon-Kamm et al., Plant Cell 2: 603 (1990). For example, resistance to glyphosate or sulfonylurea herbicides can be achieved using genes encoding mutant target enzymes, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and acetolactate synthase (ALS). can get. Resistance to glufosinate ammonium, bromoxynyl, and 2,4-dichlorophenoxyacetate (2,4-D) is a phosphinothricin acetyltransferase, nitrilase, or 2,4-dichlorophenoxyacetate that detoxifies each herbicide Obtained by using a bacterial gene encoding monooxygenase.

  Selectable marker genes of interest for the present invention include neomycin phosphotransferase II (Fraley et al., CRC Critical Reviews in Plant Science 4: 1 (1986)); cyanamide hydratase (Maier-Greiner et al., Proc. Natl. Acad. USA 88: 4250 (1991)); aspartate kinase; dihydrodipicolinate synthase (Perl et al., BioTechnology 11: 715 (1993)); bar gene (Toki et al., Plant Physiol. 100: 1503 (1992); Meagher et al., Crop Sci. 36: 1367 (1996)); tryptophan decarboxylase (Goddijn et al., Pla). t Mol.Biol.22: 907 (1993)); neomycin phosphotransferase (NEO; Southern et al., J. Mol. Appl. Gen. 1: 327 (1982)); hygromycin phosphotransferase (HPT or HYG; Shimizu et al. Mol. Cell. Biol. 6: 1074); dihydrofolate reductase (DHFR; Kwok et al., Proc. Natl. Acad. Sci. USA 83: 4552 (1986)); phosphinothricin acetyltransferase (DeBlock et al., EMBO). J. 6: 2513 (1987)); 2,2-dichloropropionic acid dehalogenase (Buchanan-Wollatron et al., J. Cell. Biochem. 13D: 330); Hydroxy acid synthase (US Pat. No. 4,761,373 to Anderson et al.); Haughn et al., Mol. Gen. Genet. 221: 266 (1988); 5-enolpyruvylshikimate phosphate synthase (aroA; Comai et al., Nature 317: 741 (1985)); haloaryl nitrilase (WO 87/04181 to Stalker et al.); Acetyl Coenzyme A carboxylase (Parker et al., Plant Physiol. 92: 1220 (1990)); dihydropteroate synthase (SulI; Guerineau et al., Plant Mol. Biol. 15: 127 (1990)); and 32 kDa Photosystem II polypeptide (PsbA; Hirschbergr et al. (1983), Science 222: 1346 (1983)).

  Gentamicin (Carler et al., Plant Mol. Biol. 17: 301-303 (1991)); Chloramphenicol (Herrera-Estrella et al., EMBO J. 2: 987 (1983)); Methotrexate ((Herrera-Estrella et al., Nature) 303: 209 (1983); Meijer et al., Plant Mol. Biol. 16: 807 (1991)); hygromycin (Waldron et al., Plant Mol. Biol. 5: 103 (1985); Zhijian et al., Plant Science 108: 219 (1995). Meijer et al., Plant Mol. Biol. 16: 807 (1991)); streptomycin (Jones et al., Mol. Gen. Genet. 210). : 86 (1987)); spectinomycin (Bretagne-Sagnard et al., Transgenic Res. 5: 131 (1996)); bleomycin (Hille et al., Plant Mol. Biol. 7: 171 (1986)); sulfonamide (Guerineau et al.). , Plant Mol.Biol. 15: 127 (1990)); Bromocykinyl (Stalker et al., Science 242: 419 (1988)); 2,4-D (Streber et al., BioTechnology 7: 811 (1989)); and phosphinothris Also included is a gene encoding resistance to Shin (DeBlock et al., EMBO J. 6: 2513 (1987)).

  The bar gene confers herbicide resistance to glufosinate herbicides such as phosphinotricin (PPT) or bialaphos. As noted above, other selectable markers that can be used in vector constructs include the pat gene, which is also for bialaphos and phosphinothricin resistance, the ALS gene for imidazolinone resistance, HPH or HYG for hygromycin resistance. Genes, ESPS synthase gene for glyphosate resistance, Hm1 gene for resistance to Hc toxin, and other selections that are used routinely and known to those skilled in the art, It is not limited to these. Yarranton, Curr. Opin. Biotech. 3: 506 (1992); Chistoperson et al., Proc. Natl. Acad. Sci. USA 89: 6314 (1992); Yao et al., Cell 71:63 (1992); Reznikoff, Mol. Microbiol. 6: 2419 (1992); Barkley et al., The Operan 177-220 (1980); Hu et al., Cell 48: 555 (1987); Brown et al., Cell 49: 603 (1987); FIGge et al., Cell 52: 713 (1988). ); Deuschle et al., Proc. Natl. Acad. Sci. USA 86: 5400 (1989); Fuerst et al., Proc. Natl. Acad. Sci. USA 86: 2549 (1989); Deuschle et al., Science 248: 480 (1990); Labow et al., Moll. Cell. Biol. 10: 3343 (1990); Zambretti et al., Proc. Natl. Acad. Sci. USA 89: 3952 (1992); Baim et al., Proc. Natl. Acad. Sci. USA 88: 5072 (1991); Wyborski et al., Nuc. Acids Res. 19: 4647 (1991); Hillenand-Wissman, Topics in Mol. And Struc. Biol. 10: 143 (1989); Degenkolb et al., Antimicrob. Agents Chemother. 35: 1591 (1991); Kleinschnid et al., Biochemistry 27: 1094 (1988); Gatz et al., Plant J. et al. 2: 397 (1992); Gossen et al., Proc. Natl. Acad. Sci. USA 89: 5547 (1992); Oliva et al., Antimicrob. Agents Chemother. 36: 913 (1992); Hlavka et al., Handbook of Experimental Pharmacology 78 (1985) and Gill et al., Nature 334: 721 (1988). Such disclosures are hereby incorporated by reference.

  The above list of selectable marker genes is not intended to be limiting. Any selectable marker gene can be used in the present invention.

  The expression cassette containing the test nucleotide sequence is transformed into duckweed plant culture, duckweed nodule culture, duckweed suspension culture or duckweed protoplast cell culture. The transformed duckweed is then regenerated and assayed to determine whether the test nucleotide sequence has the biological function of interest. It is recognized that duckweed systems can be manipulated to determine the biological function of a test nucleotide.

  In one embodiment, the biological function of interest is the ability to modulate the biological or physiological response of interest. This biological response is a response to temperature changes, response to changes in photoperiod, response to changes in water potential, response to changes in light levels, pathogen exposure (fungal pathogens, viruses, virions, nematodes, pests, and bacterial Responses to pathogens, responses to toxins (eg, high concentrations of salts, metals containing heavy metals, herbicides or other substances that interfere with important physiological functions), responses to plant hormones, responses to plant growth regulators Or any response such as a response to any other environmental disturbance.

  To identify a nucleic acid molecule of interest (eg, a nucleotide sequence encoding a polypeptide that modulates the response of interest), the response of the duckweed containing the test nucleotide sequence is assayed. In certain embodiments, a duckweed response containing the test nucleotide sequence is compared to a duckweed response not containing the test nucleotide sequence. Responses are assayed by changes in any phenotype, such as growth rate. A detectable difference in response indicates that the test nucleotide sequence is a nucleic acid molecule of interest. The nucleic acid molecule of interest (eg, modulates the response of interest) by correlating the transgenic duckweed system with a detectable difference in the biological response to the E. coli or Agrobacterium system provided as the source of test nucleotides. Can be easily identified.

  The growth rate can be determined by any method known in the art, for example, wet or dry weight, or nitrogen content. Duckweed cultures can be grown under selective or restrictive conditions. As used herein, the term “selective” or “restricted” growth conditions refers to conditions where the growth rate of untransformed duckweed is reduced compared to the growth rate under optimal conditions. Illustrative examples of selective growth conditions include restricted light levels, restricted pH conditions, or the presence of pathogens or toxins that restrict growth.

  In certain embodiments, the nucleic acid molecule of interest is a nucleotide sequence that encodes a polypeptide having a biological function of interest. The biological function of interest is any biological function, such as the ability to confer structural or mechanical properties, the ability to bind a specific substrate, the ability to bind a specific ligand, the ability to bind a specific receptor Or the ability to catalyze a particular reaction. In these instances where the polypeptide encoded by the test nucleotide sequence is secreted, the duckweed culture medium is removed to another container and the biological function of interest, such as certain structural properties, certain living It can be assayed for chemical action or ability to form a complex with a particular ligand, substrate or other agent. Once the polypeptide encoded by the test nucleotide sequence is retained in duckweed tissue, the tissue is disrupted, the protein is extracted, and the expression of the polypeptide having the biological function of interest is assayed. In certain embodiments, disruption, extraction, and / or assay steps are automated.

  In certain embodiments, the polypeptide having a biological function of interest is a variant of a naturally occurring polypeptide, and the biological function of interest is a polypeptide having a biological function of interest compared to the naturally occurring polypeptide. A change in at least one property of the peptide. Methods for making such variants are known in the art and are described elsewhere in this specification. See Example 2 in the experimental section. Properties that differ between a polypeptide with a biological function of interest (ie, a variant) and a naturally occurring polypeptide are structural properties, ability to bind a substrate, ability to bind a ligand, ability to catalyze a reaction of interest, biology Including, but not limited to, any property of interest such as the ability to function under conditions beyond the target range, or the ability to modulate a biological response. Polypeptides having properties of interest can be identified as described above.

  Each duckweed culture sample can be transformed with a nucleic acid preparation containing a sequence variant of only the original nucleotide sequence. Alternatively, each sample can be transformed with a heterologous nucleic acid preparation containing a pool of several test nucleotide sequences. About 3, about 5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 test nucleotide sequences per duckweed culture Can be used. In this method, a pool of test nucleotide sequences containing nucleotide sequences having a biological function of interest is subjected to one or more rounds of detail and screening to identify a single nucleic acid molecule of interest. The total number of test nucleotide sequences screened by any method is at least one or more, at least 10 or more, at least 100 or more, at least 1,000 or more, at least 10,000 or more, at least 100,000 or more, at least 500, 000 or more and at least 1,000,000 or more.

  In certain embodiments of the invention, a duckweed plant culture, duckweed nodule culture, duckweed suspension culture or duckweed protoplast cell culture is transformed with a reporter nucleotide sequence under conditions such that the reporter nucleotide sequence is incorporated into duckweed DNA. The nucleic acid molecule of interest is identified by conversion. The reporter nucleotide sequence includes the coding sequence for the reporter polypeptide. Reporter polypeptides are known in the art, and non-limiting examples include β-glucuronidase (GUS), luciferase, chloramphenicol acetyltransferase (CAT), or green fluorescent protein (GEP). Methods for detecting each of these reporter polypeptides are known in the art.

  In certain embodiments, the reporter nucleotide sequence lacks a transcription initiation nucleotide sequence. In another embodiment, the reporter nucleotide sequence comprises a transcription initiation nucleotide sequence. Transcription initiation nucleotide sequences are known in the art and include those described herein. In certain embodiments, the transcription initiation sequence is a minimal promoter sequence. This allows identification of the nucleotide sequence provided as a transcription enhancer when the reporter nucleotide sequence is incorporated into duckweed DNA so as to be operably linked to the enhancer sequence.

  The reporter nucleotide sequence can contain a selectable marker as described above to aid in selection of the transgenic plant. Any selectable marker known in the art can be used.

  The reporter nucleotide sequence can also include a nucleotide sequence that allows the reporter nucleotide sequence to be incorporated into duckweed DNA. In one embodiment, these nucleotide sequences are transfer DNA (T-DNA) sequences. These sequences are segments of a large Ti (tumor induction) plasmid that is harbored by all virulent Agrobacterium strains. In one embodiment, incomplete forward repeats found at the T-DNA sequence border are inserted at the 5 'and 3' ends of the reporter nucleotide sequence. The T-DNA nucleotide sequence of the present invention preferably comprises at least one T-DNA repeat sequence. See, for example, International Publication No. 93/17116 (which is incorporated herein by reference).

  Translocation of the T region into plant cells is accomplished by the vir region, another region of the Ti plasmid (Zambrski, Annu Rev. Genet. 22: 1-30 (1988); Zambrski et al., Cell 56: 193-201 ( 1989); and Baron and Zambryski, Curr. Biol. 6: 1567-1569 (1996)). The required vir region is provided in trans, for example on a second plasmid in a binary plasmid system. See, eg, Hoekema et al., Nature 310: 115-120 (1983) and Bevan, Nucl. Acids Res. 22: 8711-8721 (1984). In another embodiment, a vector containing the DNA to be transferred is introduced into Agrobacterium by triparental mating or electroporation and results in homologous recombination between the vector and the receptor Ti plasmid. .

  The claimed method provides for the identification of various nucleic acid molecules of interest. In one embodiment, the nucleic acid molecule of interest is provided as a promoter or enhancer that regulates transcription of the target nucleotide sequence. Thus, the methods of the invention can be used to identify promoters and enhancers that are involved in a biological response to a stimulus of interest. Stimuli of interest include changes in light, changes in temperature, changes in water potential, changes in day length, changes in plant hormone levels, changes in plant growth regulator levels, exposure to pathogens, and exposure to toxins However, it is not limited to these. To identify nucleic acid molecules involved in the biological response to a stimulus, a duckweed culture containing the incorporated reporter nucleotide sequence is exposed to the stimulus and the expression of the reporter polypeptide is determined. When the reporter nucleotide sequence is incorporated into duckweed DNA so as to be operably linked to a promoter or enhancer activated by stimulation, the expression of the reporter polypeptide is detected. Using a portion of the reporter nucleotide sequence as a marker, the site of incorporation of the reporter nucleotide sequence can be identified, thereby identifying a nucleotide sequence that exhibits a regulated activity in response to a stimulus.

  The methods of the invention can be used to identify nucleotide sequences that modulate transcription of target nucleotide sequences in a tissue-specific manner. Transgenic duckweed lines can be screened to identify those systems that express the reporter polypeptide only in specific duckweed tissues, such as roots, stems, flowers, seeds, or fronds. The nucleic acid molecule of interest can then be identified as described above.

  Nucleic acid molecules that regulate the translation of target nucleotide sequences (eg, reporter nucleotide sequences) include sequences that enhance transcription efficiency, eg, leader sequences located in the 5 ′ untranslated region of the target nucleotide sequence (eg, sequences that promote ribosome binding) ) And sequences that enhance mRNA stability. These nucleotide sequences may be located anywhere within the transcribed portion of the gene, such as the 5 'untranslated region of the transcript, the intron region of the gene, and the 3' untranslated region of the transcript. If the reporter nucleotide sequences are incorporated within the transcribed site of the gene, these sequences are identified. Monitor translational effects by assaying for expression of the reporter polypeptide. As described above with respect to transcriptional control sequences, nucleotide sequences that regulate translation can be controlled by specific stimuli. Accordingly, reporter polypeptide expression in response to a stimulus of interest can be monitored to identify regulatory nucleotide sequences that are activated in response to the stimulus.

  In certain embodiments, the reporter nucleotide sequence is incorporated into duckweed DNA such that its expression (ie, appendage and / or translation) is operably linked to the coding sequence of a gene that is controlled by the stimulus of interest. A chimeric polypeptide that can be transcribed and translated with a controlled gene into a nucleotide sequence encoding a reporter polypeptide and detected using the same techniques used to identify the reporter polypeptide Can be formed. Expression of the chimeric polypeptide in response to a stimulus of interest can be used to identify a transgenic duckweed system in which the reporter nucleotide sequence is operably linked to a regulated gene that is the nucleic acid molecule of interest. See Springer, Plant Cell 12: 1007-1020 (2000), which is hereby incorporated by reference.

[Experiment]
The following examples are for illustrative purposes and are not intended to limit the invention.

[Duckweed system and reproduction in high-density format]
<Use of liquid medium>
In previously described experiments, duckweed was cultured on solid media. The purpose of this experiment was to test whether duckweed can be cultured on a porous support saturated with liquid medium. This automates duckweed transformation, selection and regeneration steps, demonstrating that duckweed systems are useful for high-throughput screening of nucleotide sequences.

  A duckweed nodule culture (derived from Lemna minor C016) was transformed with the GUS expression construct using Agrobacterium-mediated transformation as follows.

  The expression vector used for the GUS expression construct was pBMSP-3. This expression vector is derived from pBMSP-1 described in US Pat. No. 5,955,646, which is hereby incorporated by reference. The pBMSP-3 transcription cassette is composed of three copies of a transcription-activating nucleotide sequence derived from Agrobacterium-tumefaciens octopine synthase, and another derived from the Agrobacterium-tumefaciens manopine synthase gene. A transcription-activating nucleotide sequence, a promoter region derived from the Agrobacterium tumefaciens manopain synthase gene, a polylinker site for insertion of a nucleotide sequence encoding a polypeptide of interest, and Agrobacterium tumefa Contains a termination sequence derived from the Ciensnopaline synthase gene. This expression vector also contains a nucleotide sequence encoding neomycin phosphotransferase II as a selectable marker. Transcription of the selectable marker sequence is driven by a promoter derived from the Agrobacterium tumefaciens nopaline synthase gene. pBMSP-3 further contains a nucleotide sequence corresponding to nucleotides 1222-1775 of the maize dehydrogenase gene (GenBank accession number X04049) inserted between the promoter and polylinker. To create the GUS expression construct used in this example, the GUS coding sequence and the kanamycin resistance gene were inserted into the pBMSP-3 expression vector.

Agrobacterium tumefaciens strain Egs05 transformed with GUS expression construct was transformed into YEB medium (1 g / l yeast extract, 5 g / l) containing 500 mg / l streptomycin, 50 mg / l spectinomycin and 50 mg / l kanamycin sulfate. Beef extract, 5 g / l peptone, 5 g / l sucrose, 0.5 g / l MgSO ₄ ).

  A duckweed nodule culture for transformation was prepared as follows. Duckweed fronds are isolated, roots are cut with a sterile scalpel, and fronds are ventral side down, 5 μM 2,4-dichlorophenoxyacetic acid, 0.5 μM 1-phenyl-3 (1,2 , 3-thiadiazol-5-yl) urea thiazulone (Sigma P6186), Murashige and Skog medium supplemented with 3% sucrose, 0.4 Difco Bacto agar (Fisher Scientific) and 0.15% Gelrite (Sigma) (catalog) No. M-5519; Sigma Chemical Corporation, St. Louis, MO) pH 5.6. The fronds were grown for 5 to 6 weeks. At this point, nodules (small yellowish cell masses) generally appeared from the central part of the ventral side. This piece of nodule tissue (average size 3-6 mg) was exfoliated from the mother frond and 3% sucrose, 0.4% Difco Bacto agar, 1 μM 2,4-dichlorophenoxyacetic acid, and 2 μM benzyladenine were added. Cultured in supplemented Murashige and Skog medium.

  A duckweed nodule culture was transformed as follows. Appropriate Agrobacterium tumefaciens strains were grown on YEB agar plates containing 50 μg / l kanamycin and 100 μM acetosyringone, and well-grown plates were supplemented with 0.6 M mannitol and 100 μM acetosyringone Resuspended in 100 ml of Murashige and Skoog medium. Auxin and cytokinin and 100 μM optimized to inoculate nodule cultures by immersing in resuspended bacterial solution for 1 to 2 minutes, blot to remove excess fluid, and promote nodule growth Plated in co-culture medium consisting of Murashige and Skoog medium supplemented with acetosyringone. The nodule culture was then incubated in the dark for 2 days.

For selection, transfer the nodule culture to an agar plate (procedure 1, see below) or US patent provisional application 60/294430 filed May 30, 2001 and filed May 28, 2002. Transferred to a 24-well plate adapted to the polyethylene support described in the US patent application entitled “Plates and Methods for High Speed Mass Processing Screening” (treatment 2, see below). To prepare a 24-well plate of polyethylene support (frit), hydrophilic polyethylene (1/16 inch thick) is cut to a size that fits the 24-well plate and drilled in the center for media change. A hole was made. These frits were autoclaved, dried and placed in a 24-well plate with the smooth surface up. The frit was wetted with 560 μl of Murashige and Skoog (pH 5.6) containing 3% sucrose, 1 μM 2,4-dichlorophenoxyacetic acid, 2 μM benzyladenine and one of the following treatments.
Treatment 1: (agar plate) 200 mg / l kanamycin sulfate and 500 mg / l cefotaxime Treatment 2: (24 well plate) 200 mg / l kanamycin sulfate and 500 mg / l cefotaxime and continuous light (20-40 μ / m ² -Incubated for 4 days. On day 4, the transformed nodule culture of treatment 1 was transferred to an agar medium containing Schenk and Hildebrandt medium with 3% sucrose, 200 mg / l kanamycin sulfate and 500 mg / l cefotaxime. For treatment 2 nodule culture, the medium was changed to 0.5X Schenk and Hildebrandt medium containing 3% sucrose, 200 mg / l kanamycin and 500 mg / l cefotaxime in 12 wells and 200 mg for the remaining 12 wells. The medium was changed to 0.5X Schenk and Hildebrandt medium containing / l kanamycin and 300 mg / l cetylpyridinium chloride. These nodule cultures were incubated under total light, and after 6 days, tissues from several samples were stained and monitored for GUS expression. The results were as follows.

  The nodule culture was further incubated for 7 weeks and monitored for duckweed frond regeneration. Results from treatment 2 wells grown with cefotaxime are shown below.

  The transformation efficiency in this experiment was at least 50%. Duckweed cultures grown on polyethylene supports produced regenerated fronds more rapidly than those grown on agar plates and transformation efficiency was higher. Thus, in addition to demonstrating that duckweed can grow in liquid media, this experiment increases the efficiency of transformation and reduces the time required to generate a transgenic duckweed line by culturing duckweed. Is shown. These findings demonstrate that duckweed systems provide a valuable method for whole plant-based screening of nucleotide sequences.

[Transformation and regeneration of duckweed in high-density format]
<Use of kanamycin as a selection marker>
Expression vectors, Agrobacterium strains, and duckweed nodule cultures in this experiment are as described in Example 1. The nodule culture used in this experiment was 2 weeks old. Co-culture was performed as described in Example 1. Murashige and Skoog medium (pH 5.6) containing 3% sucrose, 1 μM 2,4-dichlorophenoxyacetate, 2 μM benzyladenine, 500 mg / l cefotaxime and 200 mg / l kanamycin sulfate for decontamination and selection. The nodule culture was transferred to a 24-well plate adapted with a polyethylene support containing

  On the next day, the medium of the 24-well plate was changed as follows. The old medium was removed and 200 μl of fresh medium was added. Excess medium was removed. An additional 200 μl of medium was added and removed as described. Furthermore, 200 μl of medium was added. When the medium rose above the level of the polyethylene support, excess medium was removed.

  The next day, the media in the 24-well plate was changed as described above. The medium was then changed every 2 days for approximately 2 weeks. At this point, the growth of the nodule culture was monitored. Twenty of the 24 wells contained viable callus cells.

  Subsequently, the nodule cultures were maintained in 0.5X Schenk and Hildebrandt medium containing 3% sucrose, 200 mg / l kanamycin and 500 mg / l cefotaxime, and medium changes every 2 days for approximately 4 weeks and then Once a week for four more weeks. Approximately one week after the change to Schenk and Hildebrandt medium, cells from 5 wells were stained to detect GUS expression. The results are as follows.

  After 8 weeks under selection, duckweed cultures exhibited three growth patterns. 16% of the cultures exhibited rapid growth. In this case, the well was almost completely filled with duckweed fronds. 62% of the cultures show moderate growth, in which the wells are approximately half full of duckweed fronds and 12% of the samples show slow growth, in which less than half of the wells are duckweed fronds It was full.

  After 11 weeks under selection, 18 out of 24 duckweed cultures contained regenerated fronds and the overall transformation efficiency was over 75%.

[Transformation and regeneration of duckweed in high-density format]
<Use G418 as a selection marker>
The purpose of this experiment was to test methods to further increase duckweed transformation efficiency and reduce the time required to regenerate transgenic duckweed lines.

  The expression vectors and Agrobacterium strains in this example are as described in Example 1. Duckweed nodules Derived from minor strain 8627. A nodule culture was prepared substantially as described in Example 1, with the exception of some nodule tissue samples containing 3% sucrose, 0.4% Difco Bacto agar, 1 μM 2,4-dichlorophenoxyacetic acid. And cultured in Murashige and Skoog medium supplemented with 2 μM benzyladenine (referred to as “control” samples), and several nodule tissue samples were grown on 0.5X Schenk and Hildebrandt medium containing the same additives (Referred to as “pre-treatment” samples).

  Co-culture was performed as described in Example 1. For decontamination and selection, 3% sucrose, 1 μM 2,4-dichlorophenoxyacetate, 2 μM benzyladenine, 500 mg / l cefotaxime and 25 mg / l, 50 mg / l, 100 mg / l, or 200 mg / l The nodule cultures were transferred to 24-well plates adapted with a porous polyethylene support containing 560 μl per well of Murashige and Skoog medium (pH 5.6) containing various concentrations of G418.

  The duckweed medium was changed as described in Example 2. Since G418 provides a strong selection pressure, transformed cells died after 3 weeks of selection. Many regenerated duckweed fronds were observed only after 6 weeks of selection. After 8 weeks, transformation efficiency was determined as shown below.

  This example demonstrates that a transgenic duckweed line can be generated in as little as 6 weeks using the method of the present invention, with a transformation efficiency of over 80%.

[Identification of protein variants with biological functions of interest]
The following examples relate to the identification of variants of a naturally occurring polypeptide that differ from the naturally occurring polypeptide in at least one characteristic.

  To generate variants, the polypeptide coding sequence is subjected to saturation mutagenesis to create a population of nucleotide sequences, each of which encodes an amino acid variant of the originally encoded polypeptide. Such methods are known in the art, such as US Pat. Nos. 6,096,548, 6,117,679, 6,132,970, 6,165,793, and , 180, 406 (which is incorporated herein by reference).

  In order to identify nucleotide sequences encoding variants having the property of interest, a population of variant nucleotide sequences is generated as described above, cloned into an Agrobacterium binary vector, and transformed into E. coli. Approximately 5,000 to 10,000 colonies are picked from this library and the corresponding plasmid is transformed into Agrobacterium by electroporation. Automated colony harvesting techniques known in the art (eg, Uber et al., Biotechniques 11 (5): 642-647 (1991) and Watson et al., Nucleic Acids Res. 20: 4599-4606 (1992) (hereby incorporated by reference). A single Agrobacterium colony for use in duckweed transformation.

  Single colony Agrobacterium preparations in wells (1, 2, or 3 wells per Agrobacterium colony) of 24-well plates specially adapted for treatment of duckweed cultures by addition of filter support to each well In addition (see U.S. Provisional Patent Application No. 60/294430, filed May 30, 2001, and U.S. Patent Application entitled “Plates and Methods for High-Speed Mass Screening”, filed May 28, 2002), A duckweed plant culture, duckweed nodule culture, or duckweed suspension culture is then added to each well. The co-culture, selection and regeneration steps are performed in specially adapted 24-well plates as described in the previous examples. All liquid transfers are performed using automated plate processing and liquid processing procedures.

  Following regeneration, transgenic duckweed plants are assayed for expression of variant polypeptides having the property of interest. In some examples, the original encoded polypeptide contains a sequence that directs its secretion. In these cases, the media is removed from the transgenic duckweed and assayed to determine the presence of the variant polypeptide having the property of interest. Such assays are performed using high speed, high throughput screening methods known in the art.

  In those instances where the polypeptide is expressed in duckweed tissue, automated techniques are used to disrupt duckweed tissue and extract the expressed polypeptide. The extract is then assayed for the presence of a variant polypeptide having the property of interest.

  If the well containing the duckweed expressing the variant of interest contains multiple transgenic lines, the fronds from this well are separated one by one and then assayed to determine which individual line is of interest Determine whether to express a variant polypeptide with the characteristics.

[Screening of cDNA expression library in duckweed]
This example relates to screening a cDNA library for nucleotide sequences that modulate the biological response of interest.

  The cDNA expression library is transformed into Agrobacterium by electroporation and the Agrobacterium culture is derived from a single colony and maintained in a 1534 well plate. Agrobacterium transformed as described in the previous examples is co-cultured with duckweed culture. Transgenic duckweed plants are regenerated as described above and then assayed for changes in environmental disturbances such as temperature changes, day length, water potential changes, light level changes, pathogen exposure, and their response to toxin exposure. A single collection of transgenic duckweed cell lines can be used to screen for encoded proteins involved in many different responses.

  All publications and patent applications mentioned in this specification are at the level of ordinary skill in the art to which this invention pertains. All publications and patent applications are hereby incorporated by reference to the same extent as if each separate publication and patent application was specifically and individually incorporated herein. To do.

  Although the present invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

Claims (17)

A fast mass processing method for identifying nucleic acid molecules of interest, comprising:
(A) a duckweed plant culture, duckweed nodule culture, duckweed suspension culture, or duckweed protoplast cell culture on a porous support in a high-density format plate or container , comprising a sequence encoding a polypeptide Transforming with a test nucleotide sequence;
(B) a polypeptide encoded by the test nucleotide sequence on the porous support in liquid medium, wherein the duckweed plant culture, duckweed nodule culture, duckweed suspension culture, or duckweed protoplast cell culture. Culturing under conditions such that the porous support allows replacement of the liquid medium in a high density format plate or container; and
( C) determining whether the polypeptide encoded by the test nucleotide sequence modulates a biological response, wherein the test nucleotide sequence encoding the polypeptide that modulates the biological response is of interest A method, identified as a nucleic acid molecule, wherein steps (a) and (b) are performed in the same high-density format plate or container . The biological response is a response to a change in temperature, a response to a change in day length, a response to a change in water potential, a response to a change in light level, a response to exposure to a pathogen, a response to exposure to a toxin, a response to a plant hormone, or 2. The method of claim 1 selected from the group consisting of responses to plant growth regulators. A duckweed plant that has a growth rate of duckweed plant culture, duckweed nodule culture, duckweed suspension culture, or duckweed protoplast cell culture transformed with said nucleic acid molecule of interest not transformed with said nucleic acid molecule of interest The ability of the polypeptide to modulate the biological response that is altered relative to the growth rate of the culture, duckweed nodule culture, duckweed suspension culture, or duckweed protoplast cell culture The method of claim 1, as determined by growth rate determination of a duckweed plant culture, duckweed nodule culture, duckweed suspension culture, or duckweed protoplast cell culture transformed with the sequence. The growth rate of duckweed plant culture, duckweed nodule culture, duckweed suspension culture, or duckweed protoplast cell culture transformed with the nucleic acid molecule of interest under selective growth conditions is the same The growth rate of a duckweed plant culture, duckweed nodule culture, duckweed suspension culture, or duckweed protoplast cell culture not transformed with the nucleic acid molecule of interest under 4. The method of claim 3 , wherein the growth rate of duckweed plant culture, duckweed nodule culture, duckweed suspension culture, or duckweed protoplast cell culture transformed with the nucleotide sequence is determined under selective growth conditions. The growth rate of duckweed plant culture, duckweed nodule culture, duckweed suspension culture, or duckweed protoplast cell culture transformed with the nucleic acid molecule of interest under selective growth conditions is the same The growth rate of the duckweed plant culture, duckweed nodule culture, duckweed suspension culture, or duckweed protoplast cell culture not transformed with the nucleic acid molecule of interest below, wherein said test 4. The method of claim 3 , wherein the growth rate of duckweed plant culture, duckweed nodule culture, duckweed suspension culture, or duckweed protoplast cell culture transformed with the nucleotide sequence is determined under selective growth conditions. 5. The method of claim 4 , wherein the selective growth conditions comprise at least one of a high concentration of salt, a restricted light level, a restricted pH, the presence of a pathogen and the presence of a toxin. 6. The method of claim 5 , wherein the selective growth conditions comprise at least one of a high concentration of salt, a restricted light level, a restricted pH, the presence of a pathogen and the presence of a toxin.   The method of claim 1, wherein said duckweed plant culture, duckweed nodule culture, duckweed suspension culture, or duckweed protoplast cell culture is transformed by inoculation with Agrobacterium comprising said test nucleotide sequence.   The method of claim 1, wherein the duckweed plant culture, duckweed nodule culture, duckweed suspension culture, or duckweed protoplast cell culture is transformed by ballistic bombardment.   The method of claim 1, wherein the duckweed plant culture, duckweed nodule culture, duckweed suspension culture, or duckweed protoplast cell culture is transformed by electroporation. 2. The method of claim 1, wherein the duckweed protoplast cell culture is transformed by polyethylene glycol (PEG) mediated transformation.   The method of claim 1, wherein the duckweed plant culture, duckweed nodule culture, duckweed suspension culture, or duckweed protoplast cell culture is selected from the group consisting of the genus Spriodela, the genus Wolfifia, the genus Wolfiella and the genus Lemna. The duckweed plant or duckweed nodule is selected from the group consisting of Lemna minor, Lemna miniscula, Lemna aequinocitalis and Lemna gibba group 12 selected from the group of Lemna giba. 2. The method of claim 1, wherein the ability of the polypeptide to modulate the biological response is determined by a biochemical assay. 2. The method of claim 1, wherein the ability of the polypeptide to modulate the biological response is determined by a physiological assay. 2. The method of claim 1, wherein the polypeptide's ability to modulate the biological response is determined by a phenotypic change. The polypeptide that modulates the biological response has a binding site for an agent, and the presence of the polypeptide that modulates the biological response of interest comprises the polypeptide that modulates the biological response; 2. The method of claim 1 determined by detecting complex formation with said agent.